Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis

ABSTRACT

A method for detecting interactions between first and second interacting molecules a variable sensitivity. This variable sensitivity may be obtained by providing for the overexpression of either a bait hybrid protein containing a DNA binding domain (desensitization) or a prey hybrid protein containing the DNA activation domain for a reporter gene (enhanced sensitivity). The use of exogenous activators of one or the other according to the needs of a particular system is readily accomplished.

RELATED PATENT APPLICATION

[0001] This Patent Application is a Continuation of U.S. patentapplication Ser. No. 09/680,738 filed on Oct. 6, 2000, which claimspriority to U.S. Provisional Application Serial No. 60/158,079 filed onOct. 7, 1999 and incorporated by reference herein.

BACKGROUND OF THE PRESENT INVENTION

[0002] Genetically-based interaction systems are commonly used inscientific research and in commercial and therapeutic applicationsderived from that research. Current genetically-based interactionsystems are severely limited by a fixed level of interaction sensitivitywhich is either completely “on” or completely “off” (Fields and Song,1989; Bartel et al., 1993; Gyuris et al., 1993; Mendelsohn and Brent,1994; Phizicky and Fields, 1995; Bai and Elledge, 1997; Brachmann andBoeke, 1997; Finley and Brent, 1997; Young, 1998). This creates problemsrelated to both the detection of numerous biologically irrelevantinteractions, as well as a failure to detect relevant biologicalinteractions. The consequences of this problem may be either a completeinability or prolonged time required to elucidate important biologicallyrelevant interactions, cellular pathways, and potentially relatedmodulatory agents and drugs.

[0003] Historically, the first description of a genetic system to detectmolecular interactions is the two-hybrid system (Fields and Song, 1989;FIG. 1). This set forth the original concept and practice of detectingprotein-protein interactions in Saccharomyces cerevisiae. This originalsystem features detection of an in vivo protein-protein interactionwithin the nucleus of the yeast cells. These cells were engineered toexpress the visually detectable bacterial gene lacZ in the presence ofan interaction. Basically, the host cells were transformed with anexpressible gene coding for a first hybrid protein composed of a DNAbinding domain and a first polypeptide. The host cells were additionallytransformed with a second hybrid protein consisting of a transcriptionalactivation domain and a second polypeptide of stable interaction withthe first protein fragment. Finally, the cells were also transformedwith a lacZ reporter gene containing at least one DNA binding sequencefor the DNA binding domain of the first hybrid protein and capable ofbeing transcribed at increased and detectable levels when thetranscriptional activation domain of the second hybrid protein was inclose proximity. Field and Song demonstrated that when the two hybridproteins were expressed, levels of the LacZ reporter proteindramatically increased in the host cell. This indicated that the DNAbinding domain in the first hybrid protein was binding to the DNAbinding sequence of the reporter gene and that the first polypeptide ofthe first hybrid protein was interacting with the second polypeptide ofthe second hybrid protein in such a manner as to bring thetranscriptional activation domain of the second hybrid protein intoproximity of the lacZ gene and thus increase its transcription andsubsequent expression.

[0004] This basic approach has been employed in all later two-hybrid andthree-hybrid systems. Extensions of this work describe such detectioncapability in nuclear, cytoplasmic, or membrane locations of eukaryotes(Aronheim et al., 1997; Gyuris et al., 1997), as well as in prokaryotes(Bustos and Schleif, 1993; Bunker and Kingston, 1995; Hays et al.,2000). The initial art has also been subsequently extended to includemultiple prokaryotic (Bustos and Schleif, 1993; Bunker and Kingston,1995; Hays et al., 2000) and eukaryotic organisms (other fungal strains,arthropod, plant, and mammalian cells) (e.g., Vasavada et al., 1991;Fearon et al., 1992; Luo et al., 1997; Shoda et al., 2000).

[0005] Parallel approaches to genetic molecular interaction detectionhave been described for detecting protein interactions with RNA and DNA,as well as with small ligands, including peptides and drugs (Li andHerskowitz, 1993; Yang et al., 1995; SenGupta et al., 1996; Brachmannand Boeke, 1997; Young, 1998). All of these systems work on the samebasic concept of using the living cell as a means of detecting theinteraction between two intracellular molecules.

[0006] Genetic molecular detection systems following the original Fieldstwo-hybrid system also usually include the additional feature of geneticselection (Fields and Song, 1989). Selection allows the detection of aninteraction by choosing the phenotype of survival; cells containingproteins that do not interact strongly enough or at all are unable togrow, and are no longer considered. The current methods of selection arelimited to an “all or nothing” auxotrophic nutrient, antibioticselection or other means of affecting survival (Fields and Song, 1989;Gyuris et al., 1993; Bai and Elledge, 1997). Selection yields a greatadvantage for the various detection systems, since cells containingpotentially irrelevant pairs of candidate interacting molecules areeliminated without intervention from the scientist or other automatedanalysis.

[0007] However, the introduction of genetic selection introduced a newand severely limiting aspect to the in vivo genetic molecular detectionsystems. All current methods of selecting for molecular interactions invivo must make a priori assumptions about the strength of theinteractions that they detect. The systems must be constructed such thatthere is a threshold above which an interaction will be detected, andbelow which it will not. That is, there is an implicit assumption thatvery weak or transient interactions are probably less likely to be realor important. Systems are designed to exclude these interactionsbecause, if systems are too sensitive, they will detect too muchbackground. However, if the system is not sensitive at all, importantinteractions will be missed. Those constructing these systems built themand tested them, and then used the systems with the most reasonablecompromise of detection sensitivity. In short, they chose thecompositions that yielded, on average, a tolerable background whilemissing a tolerable number of biologically relevant interactions.

[0008] Early crude attempts to overcome this “all or nothing” thresholdof reporting output have included: (a) exposure of yeast to toxicnutrient analogues at sub-lethal concentrations, for example, 3-AT as ahistidine synthesis inhibitor (Mangus et al., 1998); and (b) thecreation of complicated genetic modifications of the reporter, whichgive several different fixed (nonadjustable) levels of detection (Jameset al., 1996; Finley and Brent, 1997; Serebriiskii et al., 1999). Suchcomplicated modifications include the use of (b.1.) variable numbers ofreporter binding sites, (exemplified by the use of multiple LexA bindingsites (by, e.g. multiple LexA binding sites for a Leucine reporter asdescribed in Finley and Brent, 1997), for a Leucine reporter asdescribed in Finley and Brent, 1997), and (b.2) variable distancebetween reporter binding site and the transcriptional start site (Westet al., 1984).

[0009] A feature of current detection systems is the capacity to turnthe detection of protein interactions on or off completely by providingfor the expression or lack of expression of the two-hybrid libraryfusion under standard nutrient conditions. Gyuris et al. (1993) foundthat by being able to express one of the two hybrid proteins at highlevels or by being able to limit expression of one such proteincompletely, it was possible to show in vivo that the presence of both ofthe hybrid proteins were necessary for activation of the reporter gene;in other words, they added a switch enabling on or off control of one ofthe interacting components. This control is useful and exerts itseffects by modulating reporter activity, but it does not provide for thecontinuous adjustability of the sensitivity of a two-hybrid proteininteraction system. Thus, the Gyuris system further demonstrates thelimitation of the prior art: it is either on or off, above or below thesame detection threshold set by the reporters chosen when the system wasconstructed.

[0010] The level of reporter gene expression that will result from anygiven molecule-molecule interaction in a two-hybrid system is uniformfor those molecules used in combination with that reporter. The Brentlab first demonstrated this in experiments using a traditionaltwo-hybrid protein-protein interaction system. The experiments showedthat output of the quantitative lacZ reporter was directly proportionalto the independently determined strength (or Kd) of the protein-proteininteraction for the protein fragments used in the hybrid proteins. Ifthe two proteins interacted strongly in vitro, they gave robustexpression from the two-hybrid reporters and vice versa. Therefore, theyalso demonstrated that the output of a given reporter is constant for agiven pair of interacting proteins. This is now generally accepted,since many publications of genetic molecular interactions include thequantitative reporter output from the interaction system as a relativeindication of the strength of the interaction itself (Edwards et al.,1997).

[0011] The present invention yields surprising and unexpected advantagesrelative to earlier systems in providing for adjustability of thesensitivity of such detection systems.

SUMMARY OF THE INVENTION

[0012] The present invention comprises an improved two-hybrid orthree-hybrid detection method and a kit utilizing this method. Themethod of the current invention may be used with any conventionaltwo-hybrid or three-hybrid methods, including inhibition or competitiontwo-hybrid methods, as well as any future variations of those methods.In all embodiments of the present invention, the sensitivity of adetectable reporter gene in a host cell is continuously adjustable byaltering the relative or absolute amounts of interacting moleculesprovided to the host cell. The method may be used to detect interactionsbetween any types of molecules including, but not limited to, proteins,polypeptides, DNA molecules, RNA molecules, pharmaceutical agents, otherbiological or chemical agents, and other small molecules ormacromolecules. The method may be used to detect interactions in bothprokaryotic and eukaryotic organisms or cells. The molecularinteractions may occur at various locations, including, but not limitedto, extracellular regions, the cell membrane, the cytoplasm, the nuclearmembrane, the nucleus, and other intracellular regions.

[0013] In a preferred embodiment, the first chimeric gene and the secondchimeric gene are introduced into the host cell. The host cell is thensubjected to conditions under which a first hybrid protein and a secondhybrid protein are expressed in at least sufficient quantities for thedetectable reporter gene within the host cell to be activated. The firstchimeric gene contains a first exogenously activatable promoter and asequence encoding the first hybrid protein. The first hybrid proteincontains a DNA binding domain capable of binding near the reporter geneand a first interacting polypeptide(bait). The second chimeric genecontains a second exogenously activatable promoter and a sequenceencoding a second hybrid protein. The second hybrid protein contains atranscriptional activation domain capable of inducing or increasingtranscription of the reporter gene and a second interactingpolypeptide(prey). This second polypeptide may be derived from alibrary.

[0014] The sensitivity of the reporter gene may be altered by adding afirst and/or second exogenous activator and thus, altering the relativeor absolute amounts of the first and/or second hybrid proteins. Thesealterations affect the activity and thus sensitivity of the reportergene. The sensitivity of this activation may be decreased by adding afirst exogenous activator capable of activating the first exogenouspromoter. This results in increased production of the first hybridprotein and raises its level in the host cell relative to the level ofthe second hybrid protein. Thus, after this increase, more DNA bindingsites of the reporter gene are occupied by the first hybrid proteins forwhich there is not second hybrid protein available for interaction.Therefore, less of the reporter genes are activated or activation isweaker.

[0015] The sensitivity of the reporter activation may be increased byadding a second exogenous activator capable of activating the secondexogenous promoter. This results in increased production of the secondhybrid protein and raises its level in the host cell relative to thelevel of the first hybrid protein. Thus, after this increase, more ofthe DNA binding sites of the reporter gene are occupied by a firsthybrid protein that is additionally interacting with a second hybridprotein. Therefore, more of the reporter genes are activated oractivation is stronger. Subsequent to the hybrid protein expressions,detectable reporter gene expression is measured and compared to theamount of expression in the absence of any interaction between the firsttest protein and the second test protein.

[0016] A kit utilizing the method of this invention may also beprepared. The kit may comprise any host cell described above, any firstor second chimeric genes described above, or any combination thereof.The kit may also contain the first and second exogenous activators andalso chemicals or assays for detecting the detectable reporter geneproduct.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows the Original Two-hybrid system relies on thereconstitution of a functional transcription factor to report theinteraction of two proteins, depicted as X and Y. The Fields two-hybridsystem uses the DNA-binding domain and activation domain from the Gal4ptranscription factor (figure adapted from Fields and Song, 1989).

[0018]FIG. 2 shows the Novel Library vector described herein is ashuttle vector containing ampicillin and the colE1 origin of replicationfor selection in E.coli as well as TRP1 and the 2 micron origin ofreplication for selection in yeast. Unknown cDNAs are fused to the Gal4pactivation domain, and continuously variable expression is obtained bythe induction of GRE upstream activating element(s) attached to CYC1promoter. AMP=Ampicillin, E.coli selectable marker; ori=colE1 bacterialorigin of replication; TRP=TRP1 gene, yeast selectable marker; 2um=origin of replication for yeast; GRE=Glucocortocoid Response Element;CYC1p=CYC1 promoter from yeast; Gal4AD=Gal4 activation domain;AdhT=Alcohol dehydrogenase terminator.

[0019]FIG. 3 shows the novel Bait vector in a shuttle vector containingkanamycin and the colE1 origin of replication for selection in E.coli aswell as URA3 and the 2 um origin of replication for selection in yeast.A known cDNA is fused to the Gal4p DNA binding domain, and continuouslyvariable expression is obtained by the induction of ERE element(s)attached to CYC1 promoter. KAN=Kanamycin, E.coli selectable marker;ori=colE1 bacterial origin of replication; URA=URA3 gene, yeastselectable marker; 2 um=origin of replication for yeast; ERE=EstrogenResponse Element; CYC1p=CYC1 promoter from yeast; Gal4pBD=Gal4p DNAbinding domain; AdhT=Alcohol dehydrogenase terminator.

[0020]FIG. 4 shows Continuously Dose Responsive Expression of proteinsfused to Gal4pBD in the “Bait Vector” in yHYB001 strain. Strain yHYB001with the bait vector is grown in selective minimal media with varyingconcentrations of estradiol. The bait vector contains the Gal4pBD fusedto the marker lacZ. □-gal expression assays are performed three timesper estradiol concentration; the data represents an averaging of threeassays per sample. Growth was overnight and strains were at OD₆₀₀ ca.0.8 when assayed. Strain yHYB001 is described in the text. □-galexpression assays are described in Guarente (1983). The variable “n” inthe x-axis label “Dose (10^(−n)) dexamethasone” represents any givennumber on the x-axis.

[0021]FIG. 5 shows Continuously Dose Responsive Expression of proteinsfused to Gal4pAD in the “Library Vector” in yHYB001 strain. StrainyHYB001 with the library vector is grown in selective minimal media withvarying concentrations of dexamethasone. The library vector contains theGal4pAD fused to the marker lacZ. □-gal expression assays are performedthree times per dexamethasone concentration; the data represents anaveraging of three assays per sample. Growth was overnight and strainswere at OD₆₀₀ ca. 0.8 when assayed. Strain yHYB001 is described in thetext. □-gal expression assays are described in Guarente (1983). Thevariable “n” in the x-axis label “Dose (10^(−n)) dexamethasone”represents any given number on the x-axis.

[0022]FIG. 6 shows the Principle of Variable Reporter Output withchanges in relative concentration of interactors in a novel moleculargenetic interaction detection system. The number of bars representrelative levels of library fusion protein and bait fusion proteinpresent in the cell. At equilibrium, only a fraction of the fusionproteins will be physically paired at any given time, representing theKd of the interaction (in this example, we assume 50% are bound at agiven time.) A medium sensitivity assay results when both fusionproteins are present in roughly equal amounts. At equilibrium, half ofthem are interacting, resulting in an output that is ½ of thetheoretical maximum from the reporter. This is true since half of theDNA-binding domain fusions at the reporter will not be paired to anactivating library fusion. Lower sensitivity can be achieved by reducingthe amount of library fusion and/or increasing the amount of baitfusion. A two-fold difference in levels yields, at equilibrium, ¼ of thetheoretical maximum from the reporter. High sensitivity results from anoverabundance of library fusion; a two-fold excess at equilibrium willyield an output close to the theoretical maximum for the reporter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Method to Adjust theSensitivity of Genetic Macromolecular Interaction Systems

[0023] This invention comprises novel compositions, methods and uses forcontinuously and/or discontinuously adjusting the sensitivity of geneticdetection systems to enable significantly improved detection andanalysis of molecular interactions. The molecular interactions includethose at extracellular, membrane and intracellular sites, and includebut are not limited to protein-protein interactions, protein-DNAinteractions, protein-RNA and protein-small molecule interactions ineukaryotic and prokaryotic organisms.

[0024] The method of the present invention involves changing therelative quantity of each macromolecular or small molecular componentprovided within the system, such that the absolute or relative amountsof actual interacting pairs changes within the system. By altering therelative amounts of interacting molecules (particularly the moleculebound to the detectable reporter gene), the output of the system viareporter gene expression is also altered.

[0025] In the method of the present invention, the host cell may beprovided with a detectable reporter gene. This reporter gene may beprovided before or after interacting molecules or other components areprovided. However, the reporter gene is preferably provided first sothat host cells that do not contain the reporter gene may be eliminatedbefore interacting molecules or other components are introduced. Thereporter gene may be provided through any method of gene transfercurrently known or later developed. In a preferred embodiment, thereporter gene is provided through electroporation of the host cell. Thereporter gene may also be provided in any form capable of transfer tothe host cell using the selected transfer method. For instance, it maybe provided as a portion of a plasmid.

[0026] Any detectable reporter gene that may be activated by aninteraction of the interacting molecules is appropriate for this method.For instance, the reporter gene may produce a detectable reporterprotein or other detectable gene product. The method of this inventioncan function with any detectable reporter gene because the methodinvolves changing the amounts of the interacting macromoleculesthemselves. If there are more interaction to report, any reporter genewill report this as a relatively stronger activation of the reportergene. If there are relatively fewer interactions to report, any reportersystem will report this as a relatively weaker output.

[0027] In a preferred embodiment, the reporter gene comprises at leastone DNA binding site capable of interaction with a polypeptide includinga DNA binding domain or with another DNA binding molecule, such as asmall molecule or pharmaceutical agent. This DNA binding site is locatedsuch that if a first interacting molecule binds to the site andadditionally interacts with a second interacting molecule, thetranscriptional activation domain of the second molecule will be able toactivate transcription of the reporter gene.

[0028] A first interacting molecule which may be a macromolecule orsmall molecule into a host cell or its extracellular region. Thismolecule should contain a polypeptide containing a DNA binding domain orit should contain another molecular region capable of binding DNA. Thisfirst interacting molecule may be a protein, a DNA, a RNA, or apharmaceutical agent or any other molecule that contains or may be boundto a molecule containing a DNA binding region or domain.

[0029] In a preferred embodiment, the molecule is a protein, a DNA, or aRNA. In such a preferred embodiment, the macromolecule is provided byintroducing into the host cell a first chimeric gene capable of beingtranscribed in the host cell. This first chimeric gene may include afirst exogenously activatable promoter, a sequence coding for apolypeptide, DNA, or RNA containing a DNA binding region, and a sequencecoding for the first interacting macromolecule. In a more preferredembodiment, the first chimeric gene comprises a first exogenouslyactivatable promoter and a first hybrid protein. This first hybridprotein comprises a DNA binding polypeptide and a first interactingpolypeptide (bait) capable of interacting with at least one secondinteracting polypeptide (prey).

[0030] A second interacting macromolecule or small molecule is alsointroduced into the host cell or its extracellular region. This moleculeshould contain a transcriptional activation domain. This transcriptionalactivation domain may be a polypeptide or a region of another moleculecapable of activating transcription such as a region of a pharmaceuticalagent or a nucleic acid. In a preferred embodiment, this second moleculemay be a protein, a DNA, a RNA, a pharmaceutical agent, or any othermolecule meeting the requirements stated above. In a more preferredembodiment it is a protein, a DNA, or a RNA. In such a preferredembodiment, the macromolecule may be produced in the host cell byintroducing a second chimeric gene capable of being transcribed in thehost cell. This second chimeric gene may include a second exogenouslyactivatable promoter, a sequence coding for a transcriptional activationdomain, and a sequence coding for the second macromolecule. In a morepreferred embodiment, the chimeric gene may contain a second exogenouslyactivatable promoter and a second hybrid protein. This second hybridprotein may contain a polypeptide containing a transcriptionalactivation domain and the second interacting polypeptide (prey).

[0031] In all embodiments of the present invention, the sensitivity ofthe detectable reporter gene in a host cell is continuously adjustableby altering the relative or absolute amounts of the first and/or secondinteracting molecules provided to the host cell. In a preferredembodiment, the host cell itself has the capacity to regulate theabsolute of relative levels of the first or second molecules. Asindicated in the preferred embodiments described above, this may beaccomplished by introducing chimeric genes encoding the first and secondmacromolecules and containing first and second exogenously activatablepromoters. These exogenously activatable promoters may be activated byexogenous activators.

[0032] While any promoters and activators may be used in the method ofthe present invention, in a more preferred embodiment, the activator isa natural or synthetic, metabolically active or inactive steroid,steroid analogue or mimic and the promoter induces transcription inresponse to the activator. The relative or absolute amount of at leastone of the hybrid proteins may then be controlled in a manner responsiveto the dose of one or more of these activators, its analog, or itsantagonist. Generally, if both chimeric genes are under the control ofexogenously activatable promoters, the promoters will be different foreach gene and will be activated by different molecules.

[0033] By regulating the relative levels of the first and secondinteracting molecules, it is possible to alter the sensitivity of thereporter. For instance, if the system is flooded with one component,usually the second molecule, it is possible to drive the system towardsinteraction of a first molecule bound to the reporter with a secondmolecule such that reporter activity is increased. At maximumsensitivity, every first molecule binding the reporter is involved in aninteraction with a second molecule, and therefore the reporter isactivated more often or more strongly.

[0034] It is also possible to dampen the output of the reporter byincreasing the relative amount of one of the two interacting molecules,usually the first molecule. If this is done then most of the firstmolecules bound to the reporter are not additionally bound to a secondmolecule and thus the reporter activation is lowered.

[0035] Since the strength of a given interaction between any twomolecules does not change, the capacity to regulate the relative ortotal amounts of either of the two interacting molecules results in asystem that reports interaction at continuously adjustable levels ofsensitivity. Thus, if an interaction is weak, sensitivity may beincreased by increasing the relative number of interactions. Becausethere are more interactions, the reporter will report more stronglydespite the weakness of the interactions. If an interaction is strong,sensitivity may be decreased by decreasing the relative number ifinteractions. Because there are fewer interactions, the reporter willreport less strongly. Thus, interactions that might be deemedunimportant or undetectable using a conventional two-hybrid system maybe detected with the present invention.

[0036] In another embodiment of the present invention, the cells mayadditionally be provided with other macromolecules or small moleculesthat mediate or interfere with the interaction between the first andsecond interacting molecules. For instance, in a three-hybrid system, athird macromolecule or small molecule may be provided that facilitatesor is required for the interaction of the first and second molecules.This third molecule will most commonly be a protein. It may exert itseffect by stabilizing the interaction between the first and secondmolecules or by forming a connection between them when they otherwisewould not interact.

[0037] In a preferred embodiment, this third molecule may also interferewith the interaction of the first two. For, instance, if all of themolecules are proteins, the third molecule may contain a polypeptidethat is identical or similar to the bait or prey polypeptides. Thus, thethird molecule will interfere with the ability of the bait and prey tointeract. This variation of the two-hybrid assay is commonly known as ainhibition or competition two-hybrid assay. It is especially amenable tothe method of the present invention because such assays do not currentlyprovide precise results. Thus, inhibition two-hybrid assays wouldbenefit greatly from the present invention because relative amounts ofthe bait and prey polypeptides greatly influence the ability of thethird molecule to inhibit the bait-prey interaction and thus, thesensitivity of the reporter system. The techniques for fine-tuning andvarying the amounts of the hybrid proteins of this method might also beapplied to regulate the relative or absolute amount of a thirdinhibition polypeptide in an inhibition two-hybrid assay.

[0038] The interaction between the first and second interactingmolecules may take place and be detected anywhere within the cell. Forinstance, it may occur in the nucleus, in the cytoplasm, at or in themembrane, or in an organelle. The system would be expected to worksimilarly in prokaryotes and eukaryotes, including bacteria, yeast,plant, arthropod, and mammalian cells.

[0039] In one preferred embodiment of the present invention, the methodis applied to a genetic molecular interaction detection system.Regulation of the amounts of hybrid proteins is accomplished by usingcompositions comprising alternate promoters for different intrinsiclevels of expression of a downstream hybrid protein or molecule. Thesepromoters may be derived from natural or synthetic, yeast or non-yeastsources. Regulation may be accomplished by several methods, including,but not limited to: (a) changing the promoter upstream of a hybridprotein, for instance a hybrid in which the second interacting (prey)polypeptide is derived from a library, to give different levels ofexpression, as further exemplified below, using a GAL1/10 promoter, CYC1promoter, or ADH1 promoter, which exhibit different levels of expression(Guarente and Ptashne, 1981; Guarente et al., 1982; Ammerer, 1983;Guarente, 1983; Cantwell et al., 1986); or (b) modifying the promoteritself, as further specified and exemplified below, using a GAL1/10 orCYC1 promoter (or other promoters) with the upstream activatingsequences, UAS_(G) or UAS_(C), respectively, or other activatingsequences, any of which may be positioned at various distances from thetranscriptional start sites (West et al., 1984).

[0040] In another preferred embodiment of the present invention,regulation of hybrid protein amounts is accomplished by using a singlepromoter, for example, GAL 1/10, CYC1, ADH1 or other natural orsynthetic yeast or non-yeast promoters in combination with differentupstream enhancer sequences from yeast or non-yeast sources.

[0041] In another preferred embodiment, the method of the presentinvention discloses surprising and unexpected results applicable to allknown genetic systems for detecting and analyzing protein interactionswith other proteins and with any other classes of molecules. It isclearly and categorically distinguished from the prior art, based on itssensitivity being continuously adjustable; that is, the sensitivity maybe adjusted on a plural stepped dose-responsive basis. This includes, inone preferred embodiment, pharmacologically modifying the transcriptionor expression of the fusion protein or molecule and detecting thevarious reporter gene expression levels in a single screen.

[0042] This embodiment of the present invention gives major technicaladvantages including, but not limited to: (a) detecting and analyzinginteractions of various strengths, without any prior knowledge of eventhe range of such interaction strengths; (b) avoidance of biologicallynon-relevant interactions; (c) the detection of potentially veryimportant but currently systematically undetected, weak interactions;and (d) the potential for actually quantifying the in vivo strength ofintermolecular binding, as characteristically defined by dissociationconstant (Kd) (Estojak et al., 1995). The practical implications ofthese and related advantages, include but are not limited to: (a)substantial acceleration of detecting and analyzing protein-molecularinteractions; (b) elimination of a large subset of biologicallyirrelevant but previously detected interactions; (c) detection ofbiologically important new interactions; (d) the potential for true invivo estimations and correlations of Kd; (e) substantial enhancement oflarge-scale commercial screening; and (f) substantially improvedeffectiveness and efficiency of identifying and elucidating cellularpathways, potential drug targets, potentially complementary drugs, and avariety of other scientifically and commercially important molecularinteractions.

[0043] In one preferred embodiment of the invention, an extracellularligand binds and modulates the activity of a specific transmembranereceptor to effect a dose-responsive change in expression or activity ofone or more interacting molecules. Examples of such extracellularligands include but are not limited to growth factors, cytokines,hormones, synthetic agents and biopharmaceuticals and their cellularreceptors (Mercurio and Manning, 1999; Baldwin, 1996; Mohal andSternberg, 1999).

[0044] In another preferred embodiment, an intracellular ligandinteracts either cytoplasmically or within the nucleus to modulate theexpression or activity of the interacting molecules. Examples of suchintracellular ligands include but are not limited to small molecularpharmaceutical agents and modulators, including but not limited toantimicrobial agents, anti-tumor agents, nucleic acid-binding agents,cytoskeletal active agents, chelating agents, inducers, co-repressors,and agents affecting intracellular trafficking, localization andprotection or degradation (Schena et al., 1991; Rossi and Blau, 1998).

[0045] In another preferred embodiment, a membrane-active agentinteracts to modulate the level of cellular activation or responsepotential. Examples of such an agent include but are not limited toionophores, amphoteric and hydrophobic lipid-active agents anddetergents, various anesthetics and solvents, transmembrane andintramembrane signaling agents, and farnesylating agents (Berridge etal., 1999).

[0046] In a more preferred embodiment, the amounts of the hybridproteins containing the bait and/or prey or other proteins and moleculesare continuously varied or limited using exogenously activatedpromoters, exogenous activating agents, and other molecules including,but not limited to: (i) steroid responsive elements (SRE's), includingbut not limited to those sensitive to natural or synthetic estrogens(e.g., estradiol, estrone and others), androgens, progesterones,glucocorticoids (e.g., dexamethasone, cortisone, hydrocortisone andcortisol, among others), mineralocorticoids, ecdysones, metabolicallyinactive corticoids, other steroids (e.g., ones complementary to orphanreceptors), and retinoids; and/or (ii) agonist and antagonist agents incombination with (i); (iii) any other molecules, receptors and responseelements, in any or all combinations effective to provide continuouslyvariable amounts of (iii.a) a hybrid protein containing the baitpolypeptide, (iii.b) a hybrid protein containing the prey polypeptide,which may have been derived from a library, and/or (iii.c) generally anymolecular expression involved in genetic molecular interaction systems,such as to enable the relevant detection and analysis of the precedingbiologically relevant interactions (Schena et al., 1988; Picard et al.,1990; Kralli et al., 1995; Mangelsdorf and Evans, 1995; Kliewer, 1999;Martinez et al., 1999).

[0047] Another more preferred embodiment, which forms the basis for theExamples below, comprises a novel Interaction Hybrid System (IHS) whichis steroid-hormone-dependent, continuously adjustable, and contains atraditional triple reporter in a Saccharomyces cerevisiae two-hybridsystem. This relates to and novelly extends the principles and basicdesign of a yeast two-hybrid system as first presented and patented byStan Fields (U.S. Pat. No. 5,283,173), which is incorporated byreference herein.

[0048]Saccharomyces cerevisiae strain yHYB001 was constructed containingauxotrophies for the selectable markers leu2, ade2, trp1, ura3, andarg4. The strain is deltaGAL4 and deltaGAL80, so as to enable the use ofthe GAL4 DNA-binding domain (GAL4bd) and the GAL4 transcriptionalactivation domain (GAL4ad) as fusions for two-hybrid interactiondetection, exactly as used in the original Fields two-hybrid system. Thestrain also contains integrated human estrogen receptor and integratedrat glucocorticoid receptor genes, expressed constitutively. The strainis lem1, which enables the use of decreased concentrations ofdexamethasone in yeast, presumably by eliminating a membrane pump(Kralli et al., 1995). The strain contains three integrated reportersfor the detection of two-hybrid interactions. The first is aUAS_(G)-LacZ construct for colorimetric and quantitative assays andscreening. The second and third are UAS_(G)-ADE2 and UAS_(G)-LEU2,respectively; these each enable qualitative selection for yeast thatcontain interacting hybrid proteins or other molecules based on rescueof nutrient auxotrophies.

[0049] In this embodiment, in the first hybrid protein, the bait may befused to the carboxyl-terminal end of the GAL4bd, a DNA binding domain(FIG. 3). This first hybrid protein may be transcribed in a continuousrange of amounts over up to five orders of magnitude, and under theinfluence of an estrogen response element (ERE) within a minimalpromoter. This results in variable expression of the bait first hybridprotein over a continuous range of amounts in response to changinglevels of estrogen or estrogen antagonists in the yeast growth medium.This promoter-first hybrid protein construct is provided on a two-micronplasmid either under ARG4 or URA3 selection.

[0050] The second hybrid protein may be formed by fusion of the preypolypeptide, which may be derived from a library, to thecarboxyl-terminal end of the GAL4ad, a transcriptional activation domain(FIG. 2). This second hybrid protein may be transcribed in a continuousrange of amounts over up to five orders of magnitude and under theinfluence of preferably one to six, and in the present example, three,glucocorticoid response elements (GREs) within a minimal promoter, forexample, including but not limited to that from CYC1. This results invariable expression of the second hybrid protein over a continuous rangeof amounts in response to changing levels of glucocorticoids or theirantagonists, including but not limited to dexamethasone, in the yeastgrowth medium. This promoter—second hybrid protein construct is providedon a two-micron plasmid under TRP1 selection. Both hybrid proteinplasmids are also shuttle vectors containing either ampicillin orkanamycin resistance and a colE1 origin of replication, which providefor manipulation in E. coli bacteria.

[0051] Wide ranges of estrogen and dexamethasone concentrations in theyeast medium result in wide ranges of variable and relative expressionof the hybrid proteins. Estrogen is used over a concentration range ofat least about 10⁻¹² to 10⁻⁸ M while dexamethasone is used over aconcentration range of at least about 10⁻⁷ to 10⁻⁴M. Estradiol has someeffect on yeast growth above concentrations of 10⁻⁶.

[0052] Although only preferred embodiments of the invention arespecifically described above, it will be appreciated that modificationsand variations of the invention are possible without departing from thespirit and intended scope of the invention.

[0053] The following non-limiting examples are provided to more clearlyillustrate the aspects of the invention and are not intended to limitthe scope of the invention.

EXAMPLES Example 1 Interaction Hybrid System Adjusted to Give VariableQuantitative Reporter Output without Modifying the Reporter System

[0054] As noted above, the Brent lab has shown that a given set oftwo-hybrid protein interactors yield a uniform quantitative reporteroutput directly proportional to their strength of interaction (Estojaket al., 1995). Utilizing the novel adjustable yeast interaction hybridsystem (IHS), introduced and described as a more preferred embodiment inthe paragraphs above, three sets of proteins pairs previouslydemonstrated to interact in a two-hybrid system are demonstrated to givevariable levels of reporter output when expressed at different relativeconcentrations. The level of expression of the first hybrid proteincontaining the bait is proportional to the concentration of estradiol,and the level of the second hybrid protein containing the prey derivedfrom a library is proportional to dexamethasone concentration (Kralli etal., 1995; Gaido et al., 1997 (FIGS. 4 and 5)). TABLE 1 Quantitation ofknown interactors in a traditional Two-Hybrid Screen (2HS) and the novelInteraction Hybrid System (IHS) at various levels of sensitivity LOWMEDIUM HIGH BAIT LIBRARY TRADITIONAL SENSITIVITY SENSITIVITY SENSITIVITYHYBRID HYBRID 2HS IHS IHS IHS SNF1 SNF4 300 50 250 2000 Pelle Tube 25020 150 1400 Pelle Dorsal 1300  100  1400  2500

[0055] Table 1 shows a traditional two-hybrid system and the novelInteraction Hybrid System were done using proteins previously describedin Edwards et al. (1997). Methods for analysis of the two-hybrid screenare described in Edwards et al. (1997). Low sensitivity assays in theIHS used 10⁻¹⁰M Estradiol and 10⁻⁷M Dexamethasone. Medium sensitivityassays used 10⁻¹⁰M Estradiol and 10⁻⁵M Dexamethasone. High sensitivityassays used 10⁻¹²M Estradiol and 10⁻⁴M Dexamethasone.

[0056] Table 1 demonstrates that different relative levels of expressionof a bait and of a library (prey) hybrid protein in the context of thenovel IHS system gives variable levels of reporter activity. Sinceclearly the Kd of the interaction is not changing, and the sensitivityof the reporter output has not been altered, the quantitative level ofexpression from the lacZ reporter must be altered by the change inrelative concentrations of the hybrid proteins themselves (Table 1).

[0057]FIG. 6 is an illustration of the principle of varying reporteroutput in a genetic interaction system given a constant reporter set.Yeast colonies containing identical hybrid proteins in identical strainswere observed to express reporter protein at different levels whenexposed to various steroid combinations. Yeast cells containing SNF1 andSNF4 or pelle and tube constructs, fused to the bait and prey vector(respectively, see Table 1), were plated in the corresponding minimalmedia in the presence of different concentrations of estradiol and/ordexamethasone. An integrated copy of UAS_(G)-LacZ was used as a reporterfor the interaction. UAS_(G)-LacZ expression was detected by thedevelopment of blue color in yeast colonies. Cells grown in platescontaining 10⁻¹²M estradiol and 10⁻⁴M dexamethasone showed the highestexpression of UAS_(G)-LacZ. At concentrations of 10⁻¹⁰M estradiol and10⁻⁵M dexamethasone the level of LacZ expression diminished, being atits lowest when the cells were grown at 10⁻⁹M estradiol and 10⁻⁷Mdexamethasone. The different levels of LacZ expression observedcorresponded with the sensitivity of the assay, thus at high sensitivitythe intensity of the blue color was at its maximum. As the sensitivityof the assay decreased the blue color became less intense. At theweakest level of sensitivity, a light blue color was observed. No bluecolor developed in yeast colonies containing the prey hybrid alone inthe presence 10⁻⁹M estradiol. Similarly, no blue color was observed inyeast colonies containing the bait hybrid alone in the presence of 10⁻⁴Mdexamethasone or in yeast colonies with neither prey hybrid nor baithybrid when grown at in levels of steroids sufficient to produce thehighest sensitivity level when both hybrid gene constructs were present.

Example 2 Interaction Hybrid System Adjusted to Low Sensitivity forPromiscuous Bait

[0058] When low sensitivity is desired, as in the screening of themammalian baits IRAK kinase, its Drosophila homolog Pelle kinase, or itsplant kinase homologues, high expression of the bait hybrid protein isachieved using 10⁻⁹ M estradiol (for example, as in Table 2, below). Lowrelative expression of the library/prey hybrid proteins is achieved onlyat 10⁻⁷ M dexamethasone (for example, as in Table 2, below). The excessof bait hybrid protein decreases the background expression of weak andirrelevant interactions common to these kinases (refer to FIG. 6). Thisenables the successful selection, screening and discovery of theirrespective interactions with activators and scaffolds from a random cDNAlibrary. By muting the background signal, many irrelevant interactionsare reduced or eliminated which otherwise would interfere with timelyand cost-effective analysis of these screening results. TABLE 2Low-sensitivity Screen with Pelle as bait (promiscuous bait) Total FalseBait Estradiol Dexamethasone Positives Positives Pelle  10⁻⁹M 10⁻⁷M 5-25  0-50%* Pelle 10⁻¹⁰M 10⁻⁶M 10-50 50-90% Pelle 10⁻¹¹M 10⁻⁵M 100-50090-95%** Pelle 10⁻¹²M 10⁻⁴M 10,000  >99%

[0059] Table 2 shows utilizing the novel adjustable yeast interactionhybrid system introduced and described as a more preferred embodiment inthe paragraphs above, a standard interaction assay on yeast medium ismodified to contain various concentrations of steroid substances asshown in Table 2. The readout is total positives comprising the numberof colonies surviving selection for interaction. The false positives arecolonies containing proteins not interacting with the bait Pelle asdetermined by separate in vivo or in vitro confirmation assays,including genetic analysis and immunoprecipitation. By comparing thefirst and third lines of Table 2, it is evident that a maximum of only13 false positives are obtained in the adjusted low sensitivity screen(see line 1), whereas there are at least 90 false positives observedunder screening conditions where there is no difference in expressionbetween bait and library/prey hybrid proteins (see line 3). There is noobserved decrease in sensitivity to true positives (data not shown).Note: There is no difference between lem1-1 and wild-type yeast withregard to the response of the estrogen class of steroids (Kralli et al.,1995).

Example 3 Interaction Hybrid System Adjusted to High Sensitivity forPoor Quality Bait

[0060] If high sensitivity is desired, as in the screening of themammalian baits Interleukin-1 receptor, or its Drosophila homolog Toll,or its plant or mammalian homologues, minimal expression of the baithybrid protein is achieved using 10⁻⁹M estradiol. High relativeexpression of the library/prey hybrid protein is achieved using 10⁻⁵Mdexamethasone. The excess of library/prey hybrid protein apparentlydrives the weak interaction equilibrium toward forming heterodimers;more of the limiting bait hybrid proteins are occupied at a given timeby interaction with the abundant library/prey hybrid proteins (FIG. 6).The system thereby detects the weak (high Kd) interaction of thesereceptors with their cytoskeletal adapter and cytoplasmic proteins.TABLE 3 High sensitivity Screen with toll Receptor intracellular domainas bait Total False Bait Estradiol Dexamethasone Positives PositivesToll  10⁻⁹M 10⁻⁷M 0 n/a Toll 10⁻¹⁰M 10⁻⁶M 0 n/a Toll 10⁻¹¹M 10⁻⁵M 0-50-50%* Toll 10⁻¹²M 10⁻⁴M 10-30 0-50%**

[0061] Table 3 shows using the same modified interaction hybrid systemas described above under Example 2, a standard interaction assay onyeast medium is modified to contain various concentrations of steroidsubstances. The readout is again total positives, comprising the numberof colonies surviving selection for interaction. The false positives arecolonies containing prey hybrid proteins not likely to interact with thebait Toll based on DNA sequence analysis. By comparing the third andfourth lines of Table 3, it is evident that very few positives resultfrom screens equivalent to the nonadjustable two-hybrid assayscharacteristic of the prior art (see line 3), whereas, there is asignificant increase in positives obtained from the uniquely adjustablehigh sensitivity screen of the present example (line 4). There is noobserved increase in non-specific interactions for this example.

[0062] Summarizing Examples 2 and 3, modulation, including continuousand dose-responsive modulation of the expression of bait andlibrary/prey hybrid proteins, enables the present, novel interactionhybrid system to detect both weak and strong interactions without thenecessity of changing the reporters within the system itself. Stronginteractions are not confounded by background levels, and weakinteractions are not missed entirely, as characteristically occurs whenusing standard, nonadjustable interaction hybrid systems.

Example 4 Advantages of Applying the Approach of Example 1 to Expeditethe Discovery of Novel Interactors with the Promiscuous Bait, HumanIrak1 Kinase

[0063] Example 4 demonstrates the markedly improved effectiveness andefficiency of detecting known, functionally relevant and novelinteractions with promiscuous bait, including but not limited to humanIrak1 kinase bait. Two parallel sets of screens using Irak1 as bait, areinitiated with either the Roger Brent LexA nonadjustable yeastinteraction trap (Gyuris et al., 1993) and with the present adjustableinteraction hybrid system. Using lymphocyte cDNA libraries constructedfor each system, 107 possible interactions are placed under selection.Positives are yeast colonies surviving selection and thereforecontaining putative proteins that would interact with Irak1. Results ofthe Brent system are ca. 5000 total positives, only 960 of which can beaccommodated for further analysis based on the practical limitations oftime and cost. By comparison, using the present adjustable system andsimultaneously screening at 5 levels of sensitivity, 207 yeast coloniesare selected as putative positives at low sensitivity, all of which canbe accommodated for further analysis.

[0064] Upon analysis of the 960 colonies chosen for workup from theBrent system, 48 represent multiple hits for two separate uniqueproteins known to interact with Irak1. All other putative positives arerandom and unrelated proteins designated as false positives. Bycomparison, upon analysis of all of the 207 colonies passing selectionfrom the present adjustable system, 102 represent the same two uniqueknown proteins, and importantly, two additional positives represent asingle unknown protein presumed to be rare in the cDNA library.

[0065] Screening and initial analysis using equivalent personnel andmaterials requires 4 months using the Brent system, but only 4.5 weeksusing the present adjustable system. Additionally, based on materialscost alone, the present system affords a ca. five-fold reduction inanalytical costs. A parallel five-fold reduction of personnel costs arealso achieved based on the reduction in technologist's time required foranalytical steps.

[0066] Of greatest benefit, is the elimination of random weakinteractions, enabling detection of the rare and potentially valuableunknown protein that may be involved in signal transduction ofinflammatory signal downstream of the Interleukin 1 receptor.

Example 5 Advantages of Applying the Approach of Example 2 to Expeditethe Discovery of Novel Interactors using a Poor Quality Bait

[0067] Example 5 elucidates the markedly improved effectiveness ofdetecting novel interactions with the poor quality bait, human Fc gammareceptor 1 intracellular domain (Fc gamma R1). Two parallel sets ofscreens using Fc gamma R1 as bait, are initiated with the Roger BrentLexA nonadjustable yeast interaction trap (Gyuris et al., 1993) incomparison with the present adjustable interaction hybrid system. Usinglymphocyte cDNA libraries constructed for each system, 108 possibleinteractions are placed under selection for the standard Brentinteraction system and 107 interactions are placed under selection forthe present adjustable system. Positives are yeast colonies survivingselection and therefore containing putative proteins that would interactwith Fc gamma R1. Results of the Brent system are zero positives,allowing no possibility of further analysis. By comparison, using thepresent adjustable system and simultaneously screening at 5 levels ofsensitivity, 15 yeast colonies are selected as putative positives athighest sensitivity only, all of which importantly are amenable tofurther analysis. All 15 positives represent copies of a single putativeprotein interactor, which are candidates for further biochemical andgenetic analysis as important modulators of B lymphocyte activation.

Example 6 Commercial and Scientific Relevance

[0068] The present adjustable system provides a markedly improved meansto obtain cloned DNA sequences together with their corresponding proteinstructural and functional information for new and known proteins ofknown and novel functions that can serve as candidate drugs and drugtargets. Iterative use of this system enables the improved andaccelerated elucidation of entire signaling pathways linking the cellmembrane to the nucleus for use in all scientifically and commerciallyrelevant DNA-based organisms. The increased effectiveness and efficiencyof screening for protein-protein and other relevant interactionsexhibited by the present adjustable interaction hybrid system ispotentially widely applicable to enable a markedly increased volume ofscreens per unit time and cost, as well as mass screening entailing amarkedly reduced analytical load. This results in many fewerbiologically irrelevant interactors, but retains and increases valuableand biologically important interactors. In turn, this benefitsscientific and commercial developments in the fields of medicine,pharmaceutical and biopharmaceutical discovery, agribusiness andbioinformatics, among others. Application of this improved, noveltechnology can potentially markedly enhance the bioinformation ofcellular signaling pathways, knowledge of which is becoming essential tothe rational development of drugs, antibiotics, biopharmaceuticals,diagnostics, medical interventions and agricultural products, as well asthe enhanced elucidation of gene-based disease mechanisms. Hence, thistechnology potentially provides extended benefits to diverse activities,which utilize leading-edge interaction hybrid systems both academicallyand commercially. These activities include the elucidation of genomicfunctional pathways, the rapid correlation of such information with genesequencing and induced genomic (e.g., RNA expression) assays, andacceleration of the commercial discovery and development of numerouspractical genomic products and future applications.

Example 7 Application to Other Molecular Genetic Detection Systems

[0069] The general principle of a reporter having a threshold ofdetection given a constant level of expression, transcription, orpresence for the interactors in question implies the obvious extensionof the improvements described herein to all in vivo molecular geneticdetection systems. This system would be expected to function identicallywhether the interactors are proteins, RNA, DNA, carbohydrates, smallmolecules, drugs, or other potential biological interactors. Extensionis also obvious whether the detection of an interaction occurs in thenucleus, in the cytoplasm, within the membranes, or at the membrane ofthe cell. Finally, the same modifications can be applied to prokaryoticas well as other eukaryotic organisms, including mammalian cell-basedtwo-hybrid systems.

[0070] Citations in the following list of References are incorporated inpertinent part by reference herein for the reasons cited in the text.

1. A method of detecting an interaction between a bait polypeptide and aprey polypeptide comprising: introducing a first nucleic acid encoding afirst hybrid protein into a host cell, the first nucleic acid having afirst exogenously activatable promoter, and the first hybrid proteinhaving a DNA binding region and the bait polypeptide; introducing asecond nucleic acid encoding a second hybrid protein into the host cell,the second nucleic acid having a second exogenously activatable promoterdifferent from the first exogenously activatable promoter, and thesecond hybrid protein having a transcriptional activation region and theprey polypeptide; activating the first and second promoters using firstand second exogenous activators to induce expression of the first andsecond hybrid proteins; and detecting an interaction between the baitpolypeptide and the prey polypeptide by activation of a detectablereporter gene in the host cell, wherein the DNA binding region bindsnear the reporter gene and the transcriptional activation regionactivates transcription of the reporter gene when brought into proximityto the reporter gene by an interaction between the bait polypeptide andthe prey polypeptide; wherein sensitivity of detecting an interactionmay be continuously adjusted by altering the relative or absolute amountof at least one of the first or second hybrid proteins in the host celland wherein amounts of the first and second hybrid proteins in the hostcell are independent of one another.
 2. The method of claim 1, furthercomprising continuously adjusting the amount of the first hybrid proteinin the host cell through activation of the first exogenous promoter. 3.The method of claim 1, further comprising continuously adjusting theamount of the second hybrid protein in the host cell through activationof the second exogenous promoter.
 4. The method of claim 1, wherein thefirst nucleic acid further comprises a plurality of exogenous promotersoperable to induce expression of the first hybrid protein in the hostcell over a wider continuous range of amounts than the range obtainableusing only one of the plurality of exogenous promoters.
 5. The method ofclaim 1, wherein the second nucleic acid further comprises a pluralityof exogenous promoters operable to induce expression of the secondhybrid protein in the host cell over a wider continuous range of amountsthan the range obtainable using only one of the plurality of exogenouspromoters.
 6. The method of claim 1, further comprising detecting adetectable reporter protein produced by activation of the detectablereporter gene.
 7. The method of claim 1, wherein sensitivity ofdetecting an interaction may be continuously adjusted on adose-responsive basis.
 8. The method of claim 1, further comprisinginterfering with activation of at least one of the first or secondexogenously activatable promoters by providing a modulatory agent to thehost cell.
 9. The method of claim 1, wherein at least one of the firstor second exogenous activators comprises a natural or synthetic,metabolically active or inactive steroid, steroid analogue or steroidmimic.
 10. The method of claim 1, further comprising at least one of thefirst or second exogenous activators selected from the group consistingof: cortisol, cortisone, hydrocortisone, mineralcorticoids andmineralcorticoid analogues, dexamethasone estrogen, estradiol, estrone,progesterone, androgens, ecdysone, retinoid, steroids complementary toorphan receptors, other agent operable to interact with steroidresponsive elements, and any combinations thereof.
 11. The method ofclaim 1, wherein at least one of the first or second exogenousactivators comprises a membrane-active agent or analog thereof selectedfrom the group consisting of: ionophores, anesthetic agents, detergents,amphoteric agents, hydrophobic agents, lipid-active agents, solvents,transmembrane signaling agents, intramembrane signaling agents,farnesylating agents, and any combinations thereof.
 12. The method ofclaim 1, wherein at least one of the first or second exogenousactivators comprises a small molecular pharmaceutical agent selectedfrom the group consisting of: antimicrobial agents, anti-tumor agents,nucleic acid-binding agents, cytoskeletal active agents, chelators,inducers, co-repressors, agents affecting intracellular trafficking,localization, protection and degradation of exogenous or endogenousmediators, hormones, and any combinations thereof.
 13. The method ofclaim 1, wherein at least one of the first or second exogenousactivators comprises a biomolecule or natural or syntheticbiopharmaceutical selected from the group consisting of: growth factors,cytokines, hormones, their cellular receptors, fragments thereof, mimicsthereof, and any combination thereof.
 14. A method of detecting aninteraction between a bait polypeptide and a prey polypeptidecomprising: introducing a first nucleic acid encoding a first hybridprotein into a host cell, the first nucleic acid having anestrogen-sensitive promoter, and the first hybrid protein having a GAL4binding domain and the bait polypeptide; introducing a second nucleicacid encoding a second hybrid protein into the host cell, the secondnucleic acid having a glucocorticoid-sensitive promoter, and the secondhybrid protein having a GAL4 transcriptional activation domain and theprey polypeptide; activating the promoters to induce expression of thefirst and second hybrid proteins; and detecting an interaction betweenthe bait polypeptide and the prey polypeptide by activation of aUAS_(g)-LacZ reporter gene in the host cell; wherein sensitivity ofdetecting an interaction may be continuously adjusted by altering therelative or absolute amount of at least one of the first or secondhybrid proteins in the host cell and wherein amounts of the first andsecond hybrid proteins in the host cell are independent of one another.15. The method of claim 14, wherein activating the promoters furthercomprises supplying estrogen or an estrogen analogue and aglucocoritcoid or a glucorticoid analog to the host cell.
 16. The methodof claim 14, wherein sensitivity of detecting an interaction may becontinuously adjusted by altering the amount of estrogen or an estrogenanalogue supplied to the host cell.
 17. The method of claim 14, whereinsensitivity of detecting an interaction may be continuously adjusted byaltering the amount of glucocorticoid or a glucocorticoid analoguesupplied to the host cell.
 18. The method of claim 14, furthercomprising continuously adjusting the amount of the first hybrid proteinin the host cell through activation of the estrogen-sensitive promoter.19. The method of claim 14, further comprising continuously adjustingthe amount of the second hybrid protein in the host cell throughactivation of the glucocorticoid-sensitive promoter.
 20. The method ofclaim 14, further comprising detecting LacZ produced by activation ofUAS_(g)-LacZ reporter gene.
 21. The method of claim 20, furthercomprising detecting LacZ using colorimetric analysis.